Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening
| Autor: | Evan Kleinboehl, Benjamin D. Grant, Ji Li, Tory M. Schaaf, Gregory D. Gillispie, Prachi Bawaskar, David D. Thomas, Samantha L. Yuen, Kurt C. Peterson, Ang Li |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Materials science fluorescent proteins lcsh:Biotechnology Green Fluorescent Proteins Clinical Biochemistry Biosensing Techniques biosensor 01 natural sciences Article Green fluorescent protein 03 medical and health sciences lcsh:TP248.13-248.65 SERCA Fluorescence Resonance Energy Transfer Humans time-resolved FRET drug screening small-molecule high-throughput plate reader General Medicine Fluorescence 0104 chemical sciences Microplate Reader Luminescent Proteins 010404 medicinal & biomolecular chemistry 030104 developmental biology Förster resonance energy transfer biological sciences Biophysics fluorescence Fluorescent glucose biosensor Biosensor Plate reader |
| Zdroj: | Biosensors Volume 8 Issue 4 Biosensors, Vol 8, Iss 4, p 99 (2018) |
| ISSN: | 2079-6374 |
| DOI: | 10.3390/bios8040099 |
| Popis: | We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets. |
| Databáze: | OpenAIRE |
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